The ability to analyze carbohydrates is becoming increasingly important in a variety of fields. Environmental studies often require the analysis of geochemical samples that include a complex mixture of carbohydrates. Concern over fossil fuel emissions and corresponding increases in the atmospheric carbon dioxide concentrations have stimulated study of the global carbon cycle, the geologic cycle that ultimately determines the atmospheric concentration of carbon dioxide. The response time of the carbon cycle to increased atmospheric carbon dioxide depends in large measure on the rate of preservation of detrital plant material in marine sediments. Marine sediments include a complex mixture of organic and mineral materials, including significant quantities of carbohydrates. The study of carbohydrates in geochemical samples is thus an important aspect of the study of carbon cycling.
The ability to characterize carbohydrates is also of significant importance in analytical biochemistry, and in particular to the relatively new field of glycobiology. Additional applications for carbohydrate analysis include the food sciences, biomass utilization and pulp and paper chemistry fields.
Current methods for carbohydrate analysis are complex, labor intensive and can be inaccurate. Conventional chemical methods of analysis typically include hydrolysis of the polymeric carbohydrates, i.e., polysaccharides, into the corresponding oligosaccharides and monosaccharides. These constituents can then be quantified by a variety of methods, including gas and liquid chromatography. However, the intrinsic reactivity of carbohydrates in the analytical media during hydrolysis often results in adverse reactions of the hydrolyzed mono and oligosaccharides. These reactions occur under the same conditions as required for hydrolysis. Thus, for example, during analysis of carbohydrates in marine sediments, hydrolysis may be coupled with extra-molecular condensation reactions occurring with other compounds present in the sediment, and intermolecular reactions involving rearrangements or eliminations of the saccharide moieties.
A recent improvement to conventional methods of carbohydrate depolymerization involves solvolysis in hydrogen fluoride. Solvolysis of polymeric hydrocarbons results in cleavage of glycoside linkages to form glycosyl fluorides. Hydrogen fluoride is an ideal solvolysis agent, in that solvolysis of carbohydrates that are resistant to other methods can be readily accomplished. Additionally, solvolysis with hydrogen fluoride does not result in significant decomposition of the constituent sugars, and does not affect any N-acyl substituents of acylamido sugars that may be present. Under certain conditions, O-acyl substituents that are present can also be retained. Once a carbohydrate is solvolyzed with hydrogen fluoride, the resultant glycosyl fluoride is rapidly equilibrated with oxocarbenium ion due to the stability of such ions in this medium.
Conventional methods of hydrogen fluoride solvolysis are summarized in a review article by Knirel, U.A. et al., "Application of Anhydrous Hydrogen Fluoride for the Structural Analysis of Polysaccharidcs," Advances in Carbohydrate Chemistry and Biochemistry, 47:167-202 (1989), the disclosure of which is hereby expressly incorporated by reference. However, such conventional methods have limitations associated with termination of the solvolysis reaction.
One conventional method for terminating the hydrogen fluoride solvolysis reaction is the removal of hydrogen fluoride by evaporation m vacuo. This purging of the hydrogen fluoride often results in repolymerization of the solvolyzed carbohydrate. The solvolyzed mono- and oligosaccharides undergo cross-reactions, resulting in the formation of new secondary oligosaccharides.
Other conventional methods of terminating the hydrogen fluoride solvolysis reaction involve neutralization with a suspension of calcium carbonate and dichloromethane, or precipitation with cold ether. These methods also may involve undesirable cross-reactions. The variable solubility of carbohydrates, including charged ionic carbohydrates, in ether also results in this method being less accurate than desired.
Techniques have also been developed for utilizing a solvolysis mixture of hydrogen fluoride and methanol to prepare methyl glycosides of sugars. However, when a relatively high ratio of methanol to hydrogen fluoride is utilized, the removal of the solvents from the resulting mixture by evaporation is difficult. (Knirel et al. at 174).